scholarly journals Morphological and Motility Features of the Stable Bleb-Driven Monopodial Form of Entamoeba and Its Importance in Encystation

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Deepak Krishnan ◽  
Sudip Kumar Ghosh
Keyword(s):  
Sol Gel ◽  
Leading Edge ◽  
Cell Aggregation ◽  
Time Lapse ◽  
Purine Nucleotides ◽  
Content Type ◽  
Adenosine Antagonist ◽  
Entamoeba Invadens ◽  
Directional Cell Migration

ABSTRACT Entamoeba histolytica and its reptilian counterpart and encystation model Entamoeba invadens formed a polarized monopodial morphology when treated with pentoxifylline. This morphology was propelled by retrograde flow of the cell surface resulting from a cyclic sol-gel conversion of cytoplasm and a stable bleb at the leading edge. Pentoxifylline treatment switched the unpolarized, adherent trophozoites to the nonadherent, stable bleb-driven form and altered the motility pattern from slow and random to fast, directionally persistent, and highly chemotactic. Interestingly, exogenously added adenosine produced multiple protrusions and random motility, an opposite phenotype to that of pentoxifylline. Thus, pentoxifylline, an adenosine antagonist, may be inducing the monopodial morphology by preventing lateral protrusions and restricting the leading edge to one site. The polarized form of E. invadens was aggregation competent, and time-lapse microscopy of encystation revealed its appearance during early hours, mediating the cell aggregation by directional cell migration. The addition of purine nucleotides to in vitro encystation culture prevented the formation of polarized morphology and inhibited the cell aggregation and, thus, the encystation, which further showed the importance of the polarized form in the Entamoeba life cycle. Cell polarity and motility are essential in the pathogenesis of Entamoeba parasites, and the stable bleb-driven polarized morphology of Entamoeba may also be important in invasive amoebiasis.

scholarly journals Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 880-890 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Toshiyuki Ishizuka ◽  
Shuhei Hotta ◽  
Michiko Aoki ◽  
Tomohiro Shimada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Cell Aggregation ◽  
Master Regulator ◽  
Gene Encoding ◽  
Content Type ◽  
Promoter Probe ◽  
K 12 ◽  
Specific Environmental Condition

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

scholarly journals Detection of Cell Aggregation and Altered Cell Viability by Automated Label-Free Video Microscopy

2014 ◽  
Vol 20 (3) ◽  
pp. 372-381 ◽  
Author(s):  
Obaid Aftab ◽  
Mårten Fryknäs ◽  
Ulf Hammerling ◽  
Rolf Larsson ◽  
Mats G. Gustafsson
Keyword(s):  
Cell Viability ◽  
Phase Contrast ◽  
Growth Factor Receptor ◽  
Cell Aggregation ◽  
Well Being ◽  
Time Lapse ◽  
Video Microscopy ◽  
Label Free ◽  
Monitoring Method

Automated phase-contrast video microscopy now makes it feasible to monitor a high-throughput (HT) screening experiment in a 384-well microtiter plate format by collecting one time-lapse video per well. Being a very cost-effective and label-free monitoring method, its potential as an alternative to cell viability assays was evaluated. Three simple morphology feature extraction and comparison algorithms were developed and implemented for analysis of differentially time-evolving morphologies (DTEMs) monitored in phase-contrast microscopy videos. The most promising layout, pixel histogram hierarchy comparison (PHHC), was able to detect several compounds that did not induce any significant change in cell viability, but made the cell population appear as spheroidal cell aggregates. According to recent reports, all these compounds seem to be involved in inhibition of platelet-derived growth factor receptor (PDGFR) signaling. Thus, automated quantification of DTEM (AQDTEM) holds strong promise as an alternative or complement to viability assays in HT in vitro screening of chemical compounds.

scholarly journals Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

2014 ◽  
Vol 197 (5) ◽  
pp. 819-832 ◽  
Author(s):  
Eric Bordeleau ◽  
Erin B. Purcell ◽  
Daniel A. Lafontaine ◽  
Louis-Charles Fortier ◽  
Rita Tamayo ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Cell Aggregation ◽  
Flagellar Motility ◽  
Transcription Terminator ◽  
Content Type ◽  
Type Iv ◽  
Intestinal Infections ◽  
Diguanosine Monophosphate ◽  
Primary Type

Clostridium difficileis an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration inC. difficilewas recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes,pilA1(CD3513) andpilB1(CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions.pilA1is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-roundin vitrotranscription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation inC. difficile.

scholarly journals Ethanol and Isopropanol in Concentrations Present in Hand Sanitizers Sharply Reduce Excystation of Giardia and Entamoeba and Eliminate Oral Infectivity of Giardia Cysts in Gerbils

2015 ◽  
Vol 59 (11) ◽  
pp. 6749-6754 ◽  
Author(s):  
Aparajita Chatterjee ◽  
Giulia Bandini ◽  
Edwin Motari ◽  
John Samuelson
Keyword(s):  
Giardia Duodenalis ◽  
Single Layer ◽  
Oral Route ◽  
Protozoan Parasites ◽  
Content Type ◽  
Hand Sanitizer ◽  
Amebic Dysentery ◽  
Entamoeba Invadens ◽  
Enteric Protozoan

ABSTRACTEnteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls ofGiardiaandEntamoebahave a single layer composed of fibrils of β-1,3-linked GalNAc and β-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts ofG. duodenalisand 90 to 100% of cysts ofEntamoeba invadens(a nonpathogenic model forE. histolytica), as shown by nuclear labeling with propidium iodide and failure to excystin vitro. Alcohols with or without drying collapsed the cyst walls ofGiardiabut did not collapse the cyst walls ofEntamoeba. To validate thein vitroresults, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000G. duodenaliscysts, while a commercial hand sanitizer (Purell) killedE. invadenscysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission ofGiardiaandEntamoeba.

scholarly journals Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Carrie L. Shaffer ◽  
Ellisa W. Zhang ◽  
Anne G. Dudley ◽  
Beverly R. E. A. Dixon ◽  
Kirsten R. Guckes ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Bacterial Communities ◽  
De Novo ◽  
Intracellular Survival ◽  
Purine Biosynthesis ◽  
Purine Nucleotides ◽  
Content Type ◽  
Tract Infections

ABSTRACT The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement.

scholarly journals SadA, a Trimeric Autotransporter from Salmonella enterica Serovar Typhimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

2011 ◽  
Vol 79 (11) ◽  
pp. 4342-4352 ◽  
Author(s):  
Dhaarini Raghunathan ◽  
Timothy J. Wells ◽  
Faye C. Morris ◽  
Robert K. Shaw ◽  
Saeeda Bobat ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Protective Antigen ◽  
Outer Membrane Proteins ◽  
Cell Aggregation ◽  
Sub Saharan Africa ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTSalmonella entericais a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins ofSalmonellaare of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin ofSalmonella entericaserovar Typhimurium, were examined. We demonstrated that SadA is exposed on theSalmonellacell surfacein vitroandin vivoduring infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection againstSalmonella.

scholarly journals Long-term videomicroscopy of living cells in vitro: Opportunities and prospects

2016 ◽  
Vol 14 (1) ◽  
pp. 79-91
Author(s):  
Y. I. Sheiko ◽  
N. A. Balashenko ◽  
O. V. Kvitko ◽  
I. I. Koneva ◽  
S. E. Dromashko
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Morphological Changes ◽  
Cell Activity ◽  
Cell Aggregation ◽  
Time Lapse ◽  
Video Microscopy ◽  
Culture Vessel ◽  
Anti Cancer

Aim. Intravital video microscopy of cells is a highly informative approach to the study of cell cultures. Often, this method allows refining and complementing the data obtained by researchers at the visual study of living cultures or fixed preparations. The main problem of the long intravital video microscopy is the maintenance of cell activity. To solve this problem, video-computer "Tsitomir" has been developed. Methods. During cultivation the images of the cell culture areas (from one to several hundred) specified by researcher are captured at regular intervals (time-lapse method of photography). A motorized sample stage allows moving the culture vessel with the joystick, as well as to scan the specified cell culture sites automatically. Results. In our investigations, we studied such processes as cell division, death, differentiation, motility and massive changes of cell cultures associated with cancerous transformation, including abnormal morphological changes and cell aggregation. The effectiveness of the intravital cell microscopy use to test the anti-cancer drugs is shown as well. Conclusions. Opportunities of video-complex enable its use in biomedical research, in the development of cell technologies, the study of the action of pharmacological agents and sanitary-hygienic regulation of chemicals in the cell assay systems. Obtained through "Tsitomir" photos and videos can also be used as educational material for students of biological, medical and agricultural universities.Keywords: cell culture, intravital videomicroscopy, differentiation, proliferation, anti-cancer protection.

scholarly journals Polarized Entamoeba: A model for stable bleb driven motility

2019 ◽  
Author(s):  
Deepak Krishnan ◽  
Sudip Kumar Ghosh
Keyword(s):  
Adenosine Receptor ◽  
Leading Edge ◽  
Cell Aggregation ◽  
Protozoan Parasites ◽  
Membrane Flow ◽  
Adenosine Receptor Antagonist ◽  
Intermediate Phenotypes ◽  
Adenosine Receptor Agonist ◽  
Summary Statement ◽  
Entamoeba Invadens

AbstractProtozoan parasites Entamoeba histolytica and Entamoeba invadens formed a polarized phenotype, an elongated shape with a single leading edge and a trailing edge when treated with pentoxifylline. The leading edge of the polarized morphology was a spherical protrusion devoid of F-actin but with occasional F-actin scars, indicating the presence of bleb. The polarized form was stable bleb driven since the blebbing was limited to the leading edge. Pentoxifylline induced chemokinesis in Entamoeba as it switched the motility pattern from slow and random to fast and directionally persistent. Pentoxifylline speeded up the cell aggregation in E. invadens during growth and encystation due to enhanced chemotaxis of the polarized form. The transformation of non-polarized adherent trophozoites to nonadherent stable bleb driven form occurred via lamellipodial and bleb driven adherent intermediate phenotypes. The nonadherent polarized phenotype was highly motile under confinement and moved by rearward plasma membrane flow. In contrast to pentoxifylline, adenosine, the adenosine receptor agonist, stimulated the formation of multiple protrusions leading to random motility. Thus pentoxifylline might prevent lateral protrusions by inhibiting adenosine receptor, producing the monopodial polarized morphology.Summary statementPentoxifylline, the adenosine receptor antagonist induced a stable bleb driven polarized morphology in Entamoeba characterized by fast, directionally persistent and highly chemotactic motility.

scholarly journals The movement of cell clusters in vitro: morphology and directionality

1981 ◽  
Vol 49 (1) ◽  
pp. 15-32 ◽  
Author(s):  
J. Kolega
Keyword(s):  
Neural Crest Cell ◽  
Leading Edge ◽  
Active Regions ◽  
Time Lapse ◽  
Cell Sheets ◽  
Lamellar Region ◽  
Neural Crest Cell Migration ◽  
A Cell ◽  
Crest Cell

The movement of cells in small groups, or clusters, was studied in vitro using epithelioid cells from Gordon-Kosswig melanomas (from poecelid fish) and time-lapse cinemicrography. Tumour explants cultured on glass yield cell sheets from which groups of cells separate and become independently motile clusters. These clusters typically contain 3–30 cells, but may have as many as 50. They propel themselves at speeds of 0.2-4.0 micrometer/min by means of broad hyaline lamellae. The distribution of lamellae around the perimeter of each cluster correlates with both direction and speed of cluster movement, i.e. a cluster moves with its most lamellar region at its leading edge, and the greater the extent of the leading lamellar region the greater the speed. Also, a cluster tends to keep moving in the same direction. This persistence is due to a relatively constant distribution of lamellae. Cells on the trailing edge usually lack lamellae and most are very elongate and oriented perpendicular to the direction of cluster movement. In general, whenever a cell elongates, there is a loss of lamellar activity along its taut edges, parallel to the axis of elongation. Thus, any region with less lamellar activity would tend to be elongated by the outward pull of the more active regions to either side and would, in consequence, suffer a further reduction in lamellar activity. In this way, the distribution of regions of lamellar activity is self-reinforcing and the result is persistence of movement in a particular direction. This phenomenon could play an important role in giving directionality to certain morphogenetic movements, such as neural crest cell migration.

scholarly journals Identification of Positive Chemotaxis in the Protozoan Pathogen Trypanosoma brucei

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Stephanie F. DeMarco ◽  
Edwin A. Saada ◽  
Miguel A. Lopez ◽  
Kent L. Hill
Keyword(s):  
Trypanosoma Brucei ◽  
Group Identification ◽  
Protozoan Parasite ◽  
Time Lapse ◽  
Tsetse Fly ◽  
Bacterial Colony ◽  
Content Type ◽  
Group Movement ◽  
Parasite Motility

ABSTRACT To complete its infectious cycle, the protozoan parasite Trypanosoma brucei must navigate through diverse tissue environments in both its tsetse fly and mammalian hosts. This is hypothesized to be driven by yet unidentified chemotactic cues. Prior work has shown that parasites engaging in social motility in vitro alter their trajectory to avoid other groups of parasites, an example of negative chemotaxis. However, movement of T. brucei toward a stimulus, positive chemotaxis, has so far not been reported. Here, we show that upon encountering Escherichia coli, socially behaving T. brucei parasites exhibit positive chemotaxis, redirecting group movement toward the neighboring bacterial colony. This response occurs at a distance from the bacteria and involves active changes in parasite motility. By developing a quantitative chemotaxis assay, we show that the attractant is a soluble, diffusible signal dependent on actively growing E. coli. Time-lapse and live video microscopy revealed that T. brucei chemotaxis involves changes in both group and single cell motility. Groups of parasites change direction of group movement and accelerate as they approach the source of attractant, and this correlates with increasingly constrained movement of individual cells within the group. Identification of positive chemotaxis in T. brucei opens new opportunities to study mechanisms of chemotaxis in these medically and economically important pathogens. This will lead to deeper insights into how these parasites interact with and navigate through their host environments. IMPORTANCE Almost all living things need to be able to move, whether it is toward desirable environments or away from danger. For vector-borne parasites, successful transmission and infection require that these organisms be able to sense where they are and use signals from their environment to direct where they go next, a process known as chemotaxis. Here, we show that Trypanosoma brucei, the deadly protozoan parasite that causes African sleeping sickness, can sense and move toward an attractive cue. To our knowledge, this is the first report of positive chemotaxis in these organisms. In addition to describing a new behavior in T. brucei, our findings enable future studies of how chemotaxis works in these pathogens, which will lead to deeper understanding of how they move through their hosts and may lead to new therapeutic or transmission-blocking strategies.

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